Cyclohexyl substituted taxanes and pharmaceutical compositions containing them

ABSTRACT

Taxane derivatives of the formula ##STR1## wherein R 1  is cyclohexyl, 
     R 3  is phenyl, 
     T 1  is hydrogen, hydroxyl protecting group, or --COT 2 , 
     T 2  is H, C 1  -C 6  alkyl, C 2  -C 6  alkenyl, C 2  -C 6  alkynyl or monocylic aryl, 
     Ac is acetyl, and 
     E 1  and E 2  are independently selected from hydrogen and functional groups which increase the water solubility of the taxane derivative are useful as antitumor agents.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. Ser. No.07/949,107, filed Sep. 22, 1992 now pending, which is acontinuation-in-part application of U.S. Ser. No. 07/863,849, filed Apr.6, 1992 now abandoned, which is a continuation-in-part application ofU.S. Ser. No. 07/862,955, filed Apr. 3, 1992 now abandoned, which is acontinuation-in-part of U.S. Serial No. 07/763,805, filed Sep. 23, 1991,now abandoned.

BACKGROUND OF THE INVENTION

The present invention is directed to novel taxanes which have utility asantileukemia and antitumor agents.

The taxane family of terpenes, of which taxol is a member, has attractedconsiderable interest in both the biological and chemical arts. Taxol isa promising cancer chemotherapeutic agent with a broad spectrum ofantileukemic and tumor-inhibiting activity. Taxol has a 2'R, 3'Sconfiguration and the following structural formula: ##STR2## Wherein Acis acetyl. Because of this promising activity, taxol is currentlyundergoing clinical trials in both France and the United States.

Colin et al. reported in U.S. Pat. No. 4,814,470 that taxol derivativeshaving structural formula (2) below, have an activity significantlygreater than that of taxol (1). ##STR3## R' represents hydrogen oracetyl and one of R" and R'" represents hydroxy and the other representstert-butoxycarbonylamino and their stereoisomeric forms, and mixturesthereof. The compound of formula (2) in which R" is hydroxy, R'" istert-butoxycarbonylamino having the 2'R, 3'S configuration is commonlyreferred to as taxotere.

Although taxol and taxotere are promising chemotherapeutic agents, theyare not universally effective. Accordingly, a need remains foradditional chemotherapeutic agents.

SUMMARY OF THE INVENTION

Among the objects of the present invention, therefore, is the provisionof novel taxane derivatives which are valuable antileukemia andantitumor agents.

Briefly, therefore, the present invention is directed to taxanederivatives of the formula: ##STR4## wherein

R₁ is cyclohexyl,

R₃ is phenyl,

T₁ is hydrogen, hydroxyl protecting group, or --COT₂,

T₂ is H, C₁ -C₆ alkyl, C₂ -C₆ alkenyl, C₂ -C₆ alkynyl or monocylic aryl,

Ac is acetyl, and

E₁ and E₂ are independently selected from hydrogen, hydroxy protectinggroups and functional groups which increase the water solubility of thetaxane derivative.

Other objects and features of this invention will be in part apparentand in part pointed out hereinafter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, it has been discovered thatcompounds having structural formula (3), in general, and structuralformula (4), in particular show remarkable properties, in vitro, and arevaluable antileukemia and antitumor agents. Their biological activityhas been determined in vitro, using tubulin assays according to themethod of Parness et al., J. Cell Biology, 91: 479-487 (1981) and humancancer cell lines, and is comparable to that exhibited by taxol andtaxotere. ##STR5##

Taxane (4) which has the 2'R, 3'S configuration may be obtained byreacting a β-lactam with metal alkoxides having the taxane tetracyclicnucleus and a C-13 metallic oxide substituent to form compounds having aβ-amido ester substituent at C-13. The β-lactams have the followingstructural formula: ##STR6## wherein

R₁ is cyclohexyl,

R₂ is a hydroxy protecting group, and

R₃ is phenyl.

β-lactams (5) can be prepared from readily available starting materials,as is illustrated by the following reaction scheme: ##STR7## reagents

(a) lithium diisopropyl amide, tetrahydrofuran ("THF"), -78° C. to -50°C.;

(b) lithium hexamethyl disilazane, THF, -78° C. to 0° C.;

(c) THF, -78° C. to 25° C., (2h); and

(d) triethylamine, ether, -78° C., 10 min; benzoylchloride, -78° C.

The 3-hydroxyl protecting group shown in the above reaction scheme is--SiR₅ wherein R5 is trialkyl or triaryl such as triethyl. The3-hydroxyl may be protected with other standard protecting groups suchas 1-ethoxyethyl, or 2,2,2-trichloroethoxymethyl. Additional hydroxyprotecting groups and the synthesis thereof may be found in "Protectivegroups in Organic Synthesis" by T. W. Greene, John Wiley & Sons, 1981.

The racemic β-lactams may be resolved into the pure enantiomers prior toprotection by recrystallization of the corresponding2-methoxy-2-(trifluoromethyl) phenylacetic esters. However, the reactiondescribed hereinbelow in which the β-amido ester side chain is attachedhas the advantage of being highly diastereo-selective, thus permittingthe use of a racemic mixture of side chain precursor.

The metal alkoxides having the taxane tetracyclic nucleus and a C-13metallic oxide substituent have the following structural formula:##STR8## wherein T₁ is hydrogen, hydroxyl protecting group, or --COT₂ ;T₂ is H, C₁ -C₆ alkyl, C₂ -C₆ alkenyl, C₂ -C₆ alkynyl or monocylic aryl;T₃ is hydrogen or a hydroxy protecting group; and M is a metal,preferably selected from the group comprising Group IA, Group IIA andtransition metals, most preferably, Li, Mg, Na, K or Ti.

Preferably, the metal alkoxides are prepared by reacting an alcoholhaving the taxane tetracyclic nucleus and a C-13 hydroxyl group with anorganometallic compound in a suitable solvent. Most preferably, thealcohol is a protected baccatin III, in particular, 7-O-triethylsilylbaccatin III (which can be obtained as described by Greene, et al. inJACS 110: 5917 (1988) or by other routes) or 7,10-bis-O-triethylsilylbaccatin III.

As reported in Greene et al., 10-deacetyl baccatin III is converted to7-0-triethylsilyl-10-deacetyl baccatin III according to the followingreaction scheme: ##STR9## Under what is reported to be carefullyoptimized conditions, 10-deacetyl baccatin III is reacted with 20equivalents of (C₂ H₅)₃ SiCl at 23° C. under an argon atmosphere for 20hours in the presence of 50 ml of pyridine/mmol of 10-deacetyl baccatinIII to provide 7-triethylsilyl-10-deacetyl baccatin III (8a) as areaction product in 84-86% yield after purification. The reactionproduct may then optionally be acetylated with 5 equivalents of CH₃ COCland 25 mL of pyridine/mmol of 8a at 0° C. under an argon atmosphere for48 hours to provide 86% yield of 7-O-triethylsilyl baccatin III (8b).Greene, et al. in JACS 110, 5917 at 5918 (1988).

The 7-O-triethylsilyl baccatin III (8b) is reacted with anorganometallic compound such as n-butyllithium in a solvent such astetrahydrofuran (THF), to form the metal alkoxide13-O-lithium-7-O-triethylsilyl baccatin III (9) as shown in thefollowing reaction scheme: ##STR10##

As shown in the following reaction scheme,13-O-lithium-7-O-triethylsilyl baccatin III (9) reacts with β-lactam (5)in which R₂ is triethyl silyl to provide an intermediate in which theC-7 and C-2' hydroxyl groups are protected with a triethylsilyl group.The triethyl-silyl groups are then hydrolyzed under mild conditions soas not to disturb the ester linkage or the taxane substituents.##STR11## wherein

R₁ is cyclohexyl, and

R₃ is phenyl.

Both the conversion of the alcohol to the metal alkoxide and theultimate synthesis of the taxane derivative can take place in the samereaction vessel. Preferably, the β-lactam is added to the reactionvessel after formation therein of the metal alkoxide.

Compounds of formula (1) of the instant invention are useful forinhibiting tumor growth in animals including humans and are preferablyadministered in the form of a pharmaceutical composition comprising aneffective antitumor amount of compound of the instant invention incombination with a pharmaceutically acceptable carrier or diluent.

Antitumor compositions herein may be made up in any suitable formappropriate for desired use; e.g., oral, parenteral or topicaladministration. Examples of parenteral administration are intramuscular,intravenous, intraperitoneal, rectal and subcutaneous administration.

The diluent or carrier ingredients should not be such as to diminish thetherapeutic effects of the antitumor compounds.

Suitable dosage forms for oral use include tablets, dispersible powders,granules, capsules, suspensions, syrups, and elixirs. Inert diluents andcarriers for tablets include, for example, calcium carbonate, sodiumcarbonate, lactose and talc. Tablets may also contain granulating anddisintegrating agents such as starch and alginic acid, binding agentssuch as starch, gelatin and acacia, and lubricating agents such asmagnesium stearate, stearic acid and talc. Tablets may be uncoated ormay be coated by unknown techniques; e.g., to delay disintegration andabsorption. Inert diluents and carriers which may be used in capsulesinclude, for example, calcium carbonate, calcium phosphate and kaolin.Suspensions, syrups and elixirs may contain conventional excipients, forexample, methyl cellulose, tragacanth, sodium alginate; wetting agents,such as lecithin and polyoxyethylene stearate; and preservatives, e.g.,ethyl-p-hydroxybenzoate.

Dosage forms suitable for parenteral administration include solutions,suspensions, dispersions, emulsions and the like. They may also bemanufactured in the form of sterile solid compositions which can bedissolved or suspended in sterile injectable medium immediately beforeuse. They may contain suspending or dispersing agents known in the art.

The water solubility of compounds of formula (3) may be improved bymodification of the C2' and/or C7 substituents to incorporateappropriate functional groups, E₁ and E₂. For increased watersolubility, E₁ and E₂ may independently be hydrogen and --COGCOR¹wherein

G is ethylene, propylene, --CH═CH, 1,2-cyclohexane, or 1,2-phenylene,

R¹ =OH base, NR² R³, OR³, SR³, OCH₂ CONR⁴ R^(50r), OH

R² =hydrogen, methyl

R³ =(CH₂)_(n) NR⁶ R⁷ ; (CH₂)_(n) N.sup.⊕ R⁶ R⁷ R⁸ X.sup.⊖

n=1 to 3

R⁴ =hydrogen, lower alkyl containing 1 to 4 carbons

R⁵ =hydrogen, lower alkyl containing 1 to 4 carbons, benzyl,hydroxyethyl, CH₂ CO₂ H, dimethylaminoethyl

R⁶ R⁷ =lower alkyl containing 1 or 2 carbons, benzyl or R⁶ and

R⁷ together with the nitrogen atom of NR⁶ R⁷ form the following rings##STR12##

R⁸ =lower alkyl containing 1 or 2 carbons, benzyl

X.sup.⊖=halide

base=NH₃, (HOC₂ H₄)₃ N, N(CH₃)₃, CH₃ N(C₂ H₄ OH)₂, NH₂ (CH₂)₆ NH₂,N-methylglucamine, NaOH, KOH.

The preparation of compounds in which X₁ or X₂ is --COGCOR¹ is set forthin Hangwitz U.S. Pat. No. 4,942,184 which is incorporated herein byreference.

The following examples illustrate the invention.

EXAMPLE 1 ##STR13##

To a solution of 7-triethylsilyl baccatin III (120 mg, 0.171 mmol) in1.2 mL of THF at -45° C. was added dropwise 0.104 mL of a 1.63M solutionof nBuLi in hexane. After 0.5 h at -45° C., a solution ofcis-1-benzoyl-3-triethylsilyloxy-4-(cyclohexyl)azetidin-2-one (331 mg,0.885 mmol) in 1.2 mL of THF was added dropwise to the mixture. Thesolution was warmed to 0° C. and kept at that temperature for 1 h before1 mL of a 10% solution of AcOH in THF was added. The mixture waspartitioned between saturated aqueous NaHCO₃ and 60/40 ethylacetate/hexane. Evaporation of the organic layer gave a residue whichwas purified by filtration through silica gel to give 186 mg of amixture containing(2'R,3'S)-2',7-(bis)triethylsilyl-3'-desphenyl-3'-(cyclohexyl) taxol anda small amount of the (2' S,3'R) isomer.

To a solution of 186 mg (0.171 mmol) of the mixture obtained from theprevious reaction in 11 mL of acetonitrile and 0.55 mL of pyridine at 0°C. was added 1.7 mL of 48% aqueous HF. The mixture was stirred at 0° C.for 3 h, then at 25 ° C. for 13 h, and partitioned between saturatedaqueous sodium bicarbonate and ethyl acetate. Evaporation of the ethylacetate solution gave 147 mg of material which was purified by flashchromatography to give 107 mg (73%) of 3'-desphenyl-3'-(cyclohexyl)taxol, which was recrystallized from methanol/water.

m.p.163°-164° C.;[α]²⁵ _(Na) -32.0° (c 0.0049, CHCl₃).

¹ H NMR (CDCl₃, 300 MHz) δ 8.16 (d, J=7.2 Hz, 2H, benzoateortho),7.65(d, J=7.4 Hz, 2H, benzamide ortho), 7.64-7.33(m, 6H,aromatic), 6.29(s, J=9.9 Hz, 1H, NH), 6.26 (s, 1H, H10), 6.20(dd,J=9.35, 9.35 Hz, 1H, H13), 5.68(d, J=7.7 Hz, 1H, H2β), 4.97 (d, J=7.7Hz, 1H, H5), 4.60(dd, J=4.9,1.4 Hz, 1H, H2'), 4.40(m, 1H, H7), 4.27(m,1H, H3'), 4.26(d, J=8.2 Hz, 1H, H20α), 4.21 (d, J=8.2 Hz, 1H, H20α),3.78(d, J=7.7 Hz, 1H, H3), 3.66(d, J=4.9 Hz, 1H, 2'OH), 2.58(m, 1H, H6),2.50(s, 3H, 4Ac), 2.45(d, J=3.9 Hz, 1H, 7OH), , 2.31(m, 2H, H14), 2.22(s, 3H, 10Ac), 1.86 (m, 1H, H6β), 1.83(br s, 3H, Me18), 1.75(s, 1H,1OH), 1.68 (s, 3H, Me19), 1.27-1.2(m, 11H, cyclohexyl), 1.20(s, 3H,Me17), 1.11(s, 3H, Me16).

EXAMPLE 2

Tubulin binding assays were performed using compound (4) substantiallyas set forth in Parness et al., J. Cell Biology 91: 479-487 (1981) andcompared to taxol and taxotere. The results are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                                       Tubulin Assay                                                  Compound         Init.   Rel.                                                 Name/Formula     Peak    Rate                                                 ______________________________________                                        4                172                                                          Taxol            100     98                                                   Taxotere         100     --                                                   ______________________________________                                    

EXAMPLE 4

Taxane (4) was evaluated in in vitro cytotoxicity activity against humancolon carcinoma cells HCT-116 and HCT-116/VM46. The HCT116/VM cells arecells that have been selected for teniposide resistance and express themultidrug resistance phenotype, including resistance to taxol.Cytotoxicity was assessed in HCT116 and HCT VM46 human colon carcinomacells by XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2Htetrazolium hydroxide) assay (Scudiero et al, "Evaluation of a solubletetrazolium/formazan assay for cell growth and drug sensitivity inculture using human and other tumor cell lines", Cancer Res.48:4827-4833, 1988). Cells were plated at 4000 cells/well in 96 wellmicrotiter plates and 24 hours later drugs were added and serialdiluted. The cells were incubated at 37° C. for 72 hours at which timethe tetrazolium dye, XTT, was added. A dehydrogenase enzyme in livecells reduces the XTT to a form that absorbs light at 450 nm which canbe quantitated spectrophotometrically. The greater the absorbance thegreater the number of live cells. The results are expressed as an IC₅₀which is the drug concentration required to inhibit cell proliferation(i.e. absorbance at 450 nm) to 50% of that of untreated control cells.The results are presented in Table 2. Lower numbers indicate greateractivity.

                  TABLE 2                                                         ______________________________________                                                        IC.sub.50                                                     Compound          HCT    HCT                                                  Name/Formula      116    VM46                                                 ______________________________________                                        4                 0.004  0.100                                                Taxol             0.004  0.536                                                Taxotere          0.007  0.246                                                ______________________________________                                    

In view of the above, it will be seen that the several objects of theinvention are achieved.

As various changes could be made in the above compositions withoutdeparting from the scope of the invention, it is intended that allmatter contained in the above description be interpreted as illustrativeand not in a limiting sense.

What I claim is:
 1. A taxane derivative of the formula ##STR14## whereinR₁ is cyclohexyl,R₃ is phenyl, T₁ is hydrogen, hydroxyl protectinggroup, or --COT₂, T₂ is H, C₁ -C₆ alkyl, C₂ -C₆ alkenyl, C₂ -C₆ alkynylor monocylic aryl, Ac is acetyl, and E₁ and E₂ are independentlyselected from hydrogen and hydroxyl protecting groups.
 2. A taxanederivative of the formula ##STR15## wherein R₁ is cyclohexyl,R₃ isphenyl, T₁ is hydrogen, hydroxyl protecting group, or --COT₂, T₂ is H,C₁ -C₆ alkyl, C₂ -C₆ alkenyl, C₂ -C₆ alkynyl or monocylic aryl, Ac isacetyl, and E₁ and E₂ are independently selected from hydrogen, and--COGCOR¹ wherein G is ethylene, propylene, CH═CH, 1,2-cyclohexane, or1,2-phenylene, R¹ =OH base, NR² R³, OR³, SR³, OCH₂ CONR⁴ R⁵, OH R²=hydrogen, methyl R³ =(CH₂)_(n) NR⁶ R⁷ ; (CH₂)_(n) N.sup.⊕ R⁶ R⁷ R⁸X.sup.⊖ n=1 to 3 R⁴ =hydrogen, lower alkyl containing 1 to 4 carbons R⁵=hydrogen, lower alkyl containing 1 to 4 carbons, benzyl, hydroxyethyl,CH₂ CO₂ H, dimethylaminoethyl R⁶ R⁷ =lower alkyl containing 1 or 2carbons, benzyl or R⁶ and R⁷ together with the nitrogen atom of NR⁶ R⁷form the following rings ##STR16## R⁸ =lower alkyl containing 1 or 2carbons, benzyl X.sup.⊖ =halide base=NH₃, (HOC₂ H₄)₃ N, N(CH₃)₃, CH₃N(C₂ H₄ OH)₂, NH₂ (CH₂)₆ NH₂, N-methylglucamine, NaOH, KOH.
 3. Thetaxane derivative of claim 1 wherein T₁ is --COCH₃, and E₁ and E₂ arehydrogen.
 4. The taxane derivative of claim 1 wherein the taxanederivative has the 2'R, 3'S configuration.
 5. A taxane derivative of theformula ##STR17## wherein Ph is phenyl, andAc is acetyl.
 6. Apharmaceutical composition which contains the taxane derivative of claim1 and one or more pharmacologically acceptable, inert or physiologicallyactive diluents or adjuvants.